As a recently established TTI at the NCI, only very preliminary ideas in this project can be presented: -We planned to perform chemical screenings to identify compounds able to induce quiescence in pluripotent cells such as mouse embryonic stem cells. For this, we envisioned the generation and use of three different cell line reporters: 1) A pluripotent cell line expressing a fluorescently-labelled p27-mutant form unable to interact with CDKs and only stable in quiescent cells. 2) A pluripotent cell line expressing a fluorescent reporter protein that shuttles between the nucleus and the cytoplasm based on CDK activity. 3) A pluripotent cell line with an endogenous reporter for a gene highly expressed in diapaused embryos. We finally opted for generating fluorescently-labelled p27-mutant protein mouse embryonic cell lines (mESCs) to perform the chemical screen. We used a chemical library of almost 1000 compounds, and we selected 15 compounds as potent inducers of our p27-eGFP mutant suggesting that they induced cell cycle arrest and/or quiescence. After a secondary validation screen, we are further proceeding with 9 compounds. - Using the same fluorescently-labelled p27-mutant protein we have identified the existence of endogenous quiescent cells residing within mESCs cultures. We are currently identifying the proliferation dynamics by live cell imaging and transcriptional profile by single cell RNAseq of these cells. - We are currently trying to set up similar fluorescently-labelled reporter cell lines in human embryonic stem cell lines.